The development of high through-put DNA sequencing technology, and sophisticated data-capture and computational analysis has resulted in the sequence determination of entire genomes including Drosophila melanogaster and Homo sapiens. This has identified novel “predicted” gene sequences but no associated biology ascribing function. Functional information is a prerequisite to delineate which genes may prove to be therapeutic targets for disease management and diagnosis in man.
The identification of individual gene function and the functional relationship of genes to disease states is now a pre-occupation of the Biotechnology and Pharmaceutical industry. The identification of disease related genes will allow the development of new drugs or targets for drug discovery, provide diagnostic or prognostic markers for disease and provide prescriptive guides for physicians. The latter of these will be particularly useful in diseases having complex genetics. Where genetic variation between patients can be measured, personalised medicine programs can be developed where defined patient responses to drug action are identified. The approach is expensive and time consuming, and the outcomes often subjective, lacking hard evidence relating a variation in gene expression to a functional disease related event in vivo. Validation of gene function requires studies in animal model systems which directly relate cause (i.e. a mutation in a gene sequence, a deletion or an insertion) with a measurable effect (i.e. behavioural, developmental, metabolic etc.) in the whole animal.
Gene function studies in mice and other mammals are presently restricted to:
A) Painstaking mutational analysis of individual genes in “knock-out” mice derived from libraries of embryonic stems cells (ES cells) each cell containing one or more tagged genes often introduced by viral infection.
B) The random mutation in vivo of mouse genes by alkylating agents, and subsequently whole genome sequence analysis to identify multiple mutations.
The knockout approach is valid where a function can be surmised based on sequence homology with closely related genes of known function but this approach is time consuming and labour intensive.
The alkylation approach relies entirely on whole genome sequencing to identify sites of mutation and the cataloguing of changes in previously determined behavioural traits and metabolic read-outs. Identification of a phenotypic change must then be correlated to one of perhaps one hundred alkylation events in the target mouse genome. The approach is also time consuming and requires the generation and maintenance of large mouse libraries, and is limited to inbred strains of mice (for comparative review see Abuin et al. (2002) TIB 20:36-42).
Another method for obtaining mutations is through the introduction of exogenous DNA into the genome.
Transposons are natural genetic elements capable of jumping or transposing from one position to another within the genome of a species. Mobilisation of a transposon is dependant on the expression of a transposase enzyme which binds to sequences flanking the transposon DNA leading to the excision of DNA from one position in the genome and reinsertion elsewhere in the genome. Insertion into a gene sequence will lead to a change in gene function which may, in turn, result in a measurable phenotypic change in the whole organism.
Of the three “classical” model animals, the fly, the worm and the mouse, efficient transposon based insertion methodologies have been developed for D. melanogaster and for C. elegans. The following class 2 transposon families have been identified: 1) the P family; 2) the hAt family (hobo-Ac-Tam3), (including for example hermes) and the Tcl/mariner family (including for example minos, mariner and sleeping beauty).
The introduction of P element mediated transgenesis and insertional mutagenesis in Drosophila (Spradling & Rubin (1982) Science 218:341-347) transformed Drosophila genetics and formed the paradigm for developing equivalent methodologies in other eukaryotes. However, the P element has a very restricted host range, and therefore other elements have been employed in the past decade as vectors for gene transfer and/or mutagenesis in a variety of complex eukaryotes, including nematodes, plants, mammals, fish e.g. zebrafish and birds.
The use of Drosophila P-elements in D. melanogaster for enhancer trapping and gene tagging has been described; see Wilson et al. (1989) Genes Dev 3:1301; Spradling et al. (1999) Genetics 153:135.
The hobo element of Drosophila melanogaster has been described by Gelbart W. M., Blackman R. K., (1989) Prog Nucleic Acid Res Mol Biol 36:37-46.
Hermes is derived from the common housefly. Its use in creating transgenic insects is described in U.S. Pat. No. 5,614,398, incorporated herein by reference in its entirety.
Minos, a class 2 transposon and member of the Tcl family of elements, was isolated from D. hydei and has been used for the germ line transformation of D. melanogaster, C. capitata, and Anopheles stephensi (Loukeris, T. G. et al. (1995) Proc Natl Acad Sci USA 92:9485-9; Loukeris, T. G. et al. (1995) Science 270:2002-5, Catteruccia, F. et al. (2000) Nature 405:959-962) and using transient mobilisation assays it has also been shown to be active in embryos of D. melanogaster, Aedes aegypti, Anopheles stephensi and Bombyx mori and in cell lines of D. melanogaster, Aedes aegypti, Anopheles gambiae and Spodoptera frugiperda (Catteruccia, F. et al. (2000) Proc Natl Acad Sci USA 97:2157-2162, Klinakis et al. (2000) EMBO Reports 1:416-421; Shimizu et al. (June 2000) Insect Mol Biol 9(3):277-81).
European Patent Application 0955364 (Savakis et al., the disclosure of which is incorporated herein by reference) describes the use of Minos to transform cells, plants and animals. The generation of transgenic mice comprising one or more Minos insertions is also described.
Mariner is a transposon originally isolated from Drosophila mauritiana, but since discovered in several invertebrate and vertebrate species. The use of mariner to transform organisms is described in International patent application WO99/09817.
Salmonid type transposons such as the Sleeping Beauty (SB) transposon, a Tcl/mariner-like transposable element reconstructed from fish have been described by Ivics et al. (1997) Cell 91:501-510 and Horie et al. (2001) Proc Natl Acad Sci USA 98, Issue 16, 9191-9196.
International Patent Application WO99/07871 describes the use of the Tcl transposon from C. elegans for the transformation of C. elegans and a human cell line.
PiggyBac is a transposon derived from the baculovirus host Trichplusia ni. Its use for germ-line transformation of Medfly has been described by Handler et al. (1998) PNAS (USA) 95:7520-5 and U.S. Pat. No. 6,218,185.
In the techniques described in the prior art, the use of the cognate transposase for inducing transposon jumping (or transposition) is acknowledged to be necessary.
The standard methodology for transposable element mediated transformation is by coinjecting into pre-blastoderm embryos a mixture of two plasmids: one expressing transposase (Helper) but unable to transpose, and one carrying the gene of interest flanked by the inverted terminal repeats of the element (Donor). Transformed progeny of injected animals are detected by the expression of dominant marker genes.
PCT/EP01/03341 (WO 01/71019) describes the generation of transgenic animals using transposable elements. According to this method, the transposase function is provided by crossing of transgenic organisms, one of which provides a transposon function and the other providing a transposase function in order to produce organisms containing both transposon and transposase in the required cells or tissues. The use of tissue specific chromatin opening domains directs transposase activity in a tissue specific manner and gives rise to multiple independent transposition events in somatic tissues (see Zagoraiou et al. (2001) PNAS 98:11474-11478).
Transpositions can be “tagged” allowing positional changes within complex genomes to be rapidly determined and flanking genes determined by sequence analysis. This allows an immediate link between cause (i.e. an insertional event in a specific gene or regulatory element) and effect (i.e. a phenotypic or measurable change). However, conventional methods of inducing genetic modifications by transposition suffer from the disadvantage that the tissue in which transposition has occurred will be a mosaic of individual cells each with unique transpositions. As a result, analysis of phenotype results of the transposition event may be difficult to perform as each transposition event is unique. Thus, a method of controlling a transposition event so as to provide the same genetic modification in a number of cells would provide a valuable contribution to the art.